1. Field of the Invention
The present invention relates generally to the fields of genetics and molecular biology. More particularly, it concerns the detection of nucleic acids.
2. Description of Related Art
Nucleic acid amplification and detection techniques are frequently employed in analyzing DNA samples for mutations and polymorphisms. They are also employed in the detection and typing of bacteria, viruses, and fungi, including those that are infectious pathogens, by analysis of their DNA or RNA. The most common form of nucleic acid amplification is the polymerase chain reaction (PCR), which uses short oligonucleotide sequences called primers to initiate the reaction at a specific site within the target genome. The specificity of the polymerase chain reaction is dependent upon hybridization of the primers to the target nucleic acid. Factors affecting this specificity include the melting temperature (Tm), which is the temperature at which one-half of a population of hybridized oligonucleotides will dissociate and become single stranded. Typically, a primer Tm is designed to be just above the annealing temperature set on the thermal cycling instrumentation, so that, ideally, only those target sequences which are perfectly complimentary to the primer sequences will anneal, and those which are not perfectly complimentary will not anneal. Often the temperature drops below the ideal annealing temperature, such as during PCR reactions, which include reverse transcription (RT) stages, or in the case of instrument to instrument temperature variation. These lower than ideal annealing temperatures can have adverse effects on the specificity of the PCR, allowing mis-priming events to unintended targets as well as primer-primer annealing, causing unintended amplification of primer-dimers which can out compete the intended target amplification.
Attempts have been made previously to limit the activity of the polymerase enzymes during the PCR set up and reverse transcription (RT) stages by using methods known as “hot-start.” These methods are intended to reduce the formation of primer-dimers and other products of non-specific hybridization followed by extension. In these methods, a heat activation of polymerase is used to bring the polymerase to full activity. U.S. Pat. Nos. 5,773,258 5,677,152, and 5,338,671 incorporated herein by reference use either chemical modification, or antibody binding to reduce polymerase activity prior to heat induced cleavage of chemical modifications or heat induced denaturation of the inhibiting antibody. These methods are beneficial in reducing the polymerase activity dramatically, but are known to retain varying degrees of some polymerase activity, thereby not completely eliminating the problem. The reverse transcriptase enzyme itself can also exhibit some DNA polymerase activity, which can also lead to non-specific priming events. Other hot-start methods are known as “manual hot-start” and often include withholding one or more reagents from the reaction until a later stage. These manual methods are often labor intensive, or require automated liquid handling devices, and increase the risk of contamination of the PCR reaction.
Other attempts at reducing primer-dimers and other forms of non-specific amplification include the method of using linear (non-hairpin forming) primers comprising a cleavage domain within the target binding region as well as a blocking group at the 3′ end of each primer. These methods also claim the use of a hotstart cleaving enzyme which is thermostable. Examples are described in US2012/0258455 and US2013/0288245, both of which are incorporated herein by reference. While these methods may provide some increased level of specificity, they suffer from the same drawbacks as other hot-start methods, in that some enzyme activity remains in these methods prior to heat activation.
Kaboev et. al. 2000, described using hairpin-like structured primers, to achieve an increase in specificity. This method is restrictive however in the practical design of PCR primer-based assays, in that the developer is limited to using natural target sequences for the oligonucleotide composition of the stem.
There remains a need to provide a method of priming and amplification that provides superior prevention of primer-dimers and other artifacts of false priming events, particularly when the reaction is exposed to lower than ideal annealing temperatures.